Passage through the eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). The activity of CDKs is regulated at a number of levels, including binding of inhibitor proteins. These inhibitor proteins are critical for tumor suppression and growth control. For example, mutations in CDK inhibitor proteins have been found in a number of human cancers. Furthermore, mice deleted for CDK inhibitors show an increased frequency in tumor formation or decreased tissue differentiation. In the yeast, Saccharomyces cerevisiae, Sic1p functions as an inhibitor of the CDK complexes involved in S phase and progression through mitosis. Although Sic1p is not essential, strains deleted for sic1 demonstrate an increased frequency of chromosome breakage and loss. To understand the requirement for CDK inhibition at the G1 to S phase boundary and during the exit from mitosis, mutations that were lethal in combination with deletion of sic1 were isolated. Once such mutation is sid2-1. Preliminary characterization of sid2 alleles suggests the protein is required for DNA replication or repair. To determine more precisely the role of Sid2p function, DNA synthesis will be analyzed in sid2 mutant strains. Specifically, origin firing, DNA elongation and Okazaki fragment maturation will .be assayed to determine if there are defects in sid2 cells. If a defect is identified, mutations in other genes causing similar defects will be analyzed to see if they also affect viability in the absence of the CDK inhibitor SIC1. This may allow elucidation of the role of CDK regulation at the G1/S phase transition. Further analysis of the role of SID2 will focus on molecular, genetic and biochemical characterization of genes identified as suppressors or enhancers of sid2 defects. One such gene encodes the essential DNA replication protein, Cdc6p. The region of Cdc6p required for these interactions will be determined and physical interactions between Sid2p and Cdc6p will be tested directly. A number of dominant suppressors of a temperature sensitive sid2 allele have been isolated. These will be cloned and characterized. The identity of these proteins may help illuminate the role of Sid2p and could also identify new proteins required for DNA replication or repair. Finally, the requirement for various checkpoint and repair pathways will be analyzed to determine the molecular basis of the defect in sid2 mutant cells.